Novel disubstituted adamantyl derivative or pharmaceutically acceptable salt thereof, production method for same, and pharmaceutical composition for suppressing cancer metastasis comprising same as active ingredient

ABSTRACT

The present invention relates to a novel disubstituted adamantyl derivative or the pharmaceutically acceptable salts thereof, a method for preparing the same, and a pharmaceutical anticancer or antimetastasis composition comprising the same as an active ingredient. The disubstituted adamantyl derivative of the present invention suppressed accumulation of HIF-1α, inhibiting the expression of the metastasis related protein Twist dose-dependently. Thus, the disubstituted adamantyl derivative of the invention is effective in inhibiting the expressions of the metastasis related proteins, β-catenin and RohA, and the EMT related genes such as MMP2 and MMP9, without cytotoxicity. Therefore, the disubstituted adamantyl derivative or the pharmaceutically acceptable salts thereof of the invention can be efficiently used as a pharmaceutical anticancer or antimetastasis composition.

CROSS-REFERENCES TO RELATED APPLICATION

This patent application claims the benefit of priority from KoreanPatent Application No. 10-2012-0137902, filed on Nov. 30, 2012.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel disubstituted adamantylderivative or a pharmaceutically acceptable salt thereof, a method forproducing the same, and a pharmaceutical composition for inhibitingcancer metastasis containing the same as an active ingredient

2. Description of the Related Art

Even after all the efforts over the past tens of years, cancer is stillone of the incurable diseases. The reason why cancer is so difficult totreat is because cancer cells progress malignantly which favors thecancer cell survival and thus they invade in surrounding tissues andeventually move to other organs, that is so called metastasis. Cancermetastasis is a critical reason of death among cancer patients.Approximately ⅓ of patients diagnosed as cancer already have metastaticcancer at the time of diagnosis. Another ⅓ of cancer patients alsodisplay a minor or early stage of metastasis even though that is tooearly to be detected by diagnostic examinations, so that they have agreat potential of having metastatic cancer when they are only treatedlocally for their primary cancers. That is, metastatic cancer isdeveloped from primary cancer actually in ⅔ of cancer patients.Therefore, an efficient treating agent that can prevent and at the sametime control cancer metastasis after surgical operation is urgentlyrequired.

Cancer metastasis is accomplished by the steps of adhesion, invasion,and angiogenesis. In the step of adhesion, angiopoietin-2,angiopoietin-like-4, Cox-2, MMP-1, MMP-2, MMP-3, MMP-10, PGF, and VEGFcan weaken the binding between the vascular cells in the tissue wherethe cancer cell would want to invade in order for the cancer cells tomove in easily from the blood vessel to that tissue, that is they makeextravasation easy. Metastasis can be explained by the process ofEMT/MET (Epithelial-Mesenchymal Transition/Mesenchymal-EpithelialTransition) which is composed of the following steps; epithelial cellsare converted into mesenchymal cells and the converted mesenchymal cellsmigrate through blood vessels and are landed in another organ; and thenthe landed mesenchymal cells are converted reversely into epithelialcells. At this time, such genes as Twist, SNAIL, and ID1 induce EMT andaccelerate metastasis by endowing cancer stem cell like characteristicsthereto.

In the meantime, HIF-1 is the most important molecule in regulating theadaptation of cancer cell under hypoxia. Particularly, the level ofHIF-1α protein is closely related to the prognosis of cancer patients.When cancer cells are under hypoxia, the cells induce HIF-1αaccumultion, or the mentioned growth factors above can also induceHIF-1α activation. Also, the activation of HIF-1α can be induced by theactivation of an oncogene or the inactivation of a tumor suppress genelike pVHL. The activated HIF-1α induces the expressions of hexokinase 2,glucose transporter 1, erythropoietin, IGF-2, endoglin, VEGFA, MMP-2,MMP-9, uPAR, and MDR1, by which cancer cells acquire suchcharacteristics as resistance against apoptosis, promoted angiogenesis,promoted cell proliferation, cell migration, metastasis, invasion, etc,resulting in the malignance of cancer.

HIF-1 also plays an important role in regulating EMT(epithelial-mesenchymal transition) related genes in the course ofmetastasis. HIF-1 reduces E-cadherin, but increases the expressions offibronectin, vimentin, and Twist, suggesting that HIF-1 can promotemetastasis particularly the stage of EMT. Twist not only plays animportant role in gastrulation and mesoderm formation but also increasesthe expression of metastasis related proteins such as β-catenin andRhoA, etc, so that it has been recognized as a crucial factor forinducing EMT and cell migration (Cell Cycle, 2008, 7. 14, 2090-2096).

Up to date, studies have been actively going on to develop an anticanceragent inhibiting metastasis and also suppressing cancer stem cells byusing EMT promoting transcription factors. As a result, numbers ofanticancer agents such as Taxol, rapamycin, and 17-AAG(17-allylaminogeldanamycin) have been developed. These anticancer agentsare to inhibit the functions of adhesion molecules including integrinfamily mainly expressed on the surface of cancer cells, which areexemplified by extracellular matrix components like vitronectin,laminin, and fibronectin, or to suppress MMP and type IV collagenase toinhibit metastasis (Cancer Research, 53, 2087-2091, 1993). However, themethod to inhibit metastasis by using the conventional anticancer agentsis only effective in inhibiting the invasion into other organs of cancercells that have already traveled through blood stream from the organwhere the cancer cells have been originally proliferated. Therefore,this conventional method is not the fundamental treatment method.

In the course of study to find out a compound that can suppress theaccumulation of HIF-1α, the present inventors confirmed that theinhibition of HIF-1α resulted in the suppression of Twist expression,leading to the completion of this invention.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a noveldisubstituted adamantyl derivative.

It is another object of the present invention to provide a method forpreparing the disubstituted adamantyl derivative.

It is also an object of the present invention to provide apharmaceutical composition for the inhibition of metastasis comprisingthe disubstituted adamantyl derivative or the pharmaceuticallyacceptable salts thereof as an active ingredient.

It is further an object of the present invention to provide apharmaceutical anticancer composition comprising the disubstitutedadamantyl derivative or the pharmaceutically acceptable salts thereof asan active ingredient.

To achieve the above objects, the present invention provides the noveldisubstituted adamantyl derivative represented by the below formula 1 orthe pharmaceutically acceptable salts thereof:

(In the formula 1, R¹ and R² are as defined in this description).

The present invention also provides a method for preparing thedisubstituted adamantyl derivative represented by formula 1 wherein thecompound represented by formula 2 is reacted with the compoundrepresented by formula 3 to give the compound represented by formula 1,as shown in the below reaction formula 1.

(In the reaction formula 1, R¹ and R² are as defined in thisdescription).

Further, the present invention provides a pharmaceutical composition forthe inhibition of metastasis comprising the disubstituted adamantylderivative represented by formula 1 or the pharmaceutically acceptablesalts thereof as an active ingredient.

(In the formula 1, R¹ and R² are as defined in this description).

The present invention also provides a pharmaceutical anticancercomposition comprising the disubstituted adamantyl derivativerepresented by formula 1 or the pharmaceutically acceptable saltsthereof as an active ingredient.

(In the formula 1, R¹ and R² are as defined in this description).

Advantageous Effect

The disubstituted adamantyl derivative of the present invention not onlyinhibits metastasis-related HIF-1 and accordingly inhibits theexpression of metastasis-related protein Twist, β-catenin and RohA andthe EMT related genes such as MMP2 and MMP9, suggesting that it isexcellent in inhibiting cancer metastasis. In addition, thedisubstituted adamantyl derivative of the present invention does notdisplay side effects attributed to cytotoxicity when it is absorbed in aliving body, suggesting as an efficient pharmaceutical anticancercomposition or antimetastasis composition.

BRIEF DESCRIPTION OF THE DRAWINGS

The application of the preferred embodiments of the present invention isbest understood with reference to the accompanying drawings, wherein:

FIG. 1 is a graph illustrating the inhibition of HRE activity accordingto the concentrations of the compounds prepared in Examples 1 and 2.

FIG. 2 is a photograph illustrating the inhibitory effect of thecompound prepared in Example 2 according to the concentration thereof onthe accumulation of HIF-1α under hypoxia.

FIG. 3 is a photograph illustrating the inhibitory effect of thecompound prepared in Example 2 on the transcription of the metastasisrelated genes such as MMP2, MMP9, and uPA by suppressing HIF-1 accordingto the concentration.

FIG. 4 is a photograph illustrating the inhibitory effect of thecompound prepared in Example 2 according to the concentration thereof onthe expression of the EMT promoting gene including Twist under hypoxia.

FIG. 5 is a photograph illustrating the inhibition of cancer cellmigration in the group treated with the compound of Example 2.

FIG. 6 is a graph illustrating the time-dependent cancer cell migrationobserved in the group treated with the compound of Example 2.

FIG. 7 is a photograph illustrating the inhibition of cancer cellinvasion in the group treated with the compound of Example 2.

FIG. 8 is a graph illustrating the tumor volume changes over the timeafter the administration of the compound of Example 2.

FIG. 9 is a photograph illustrating the inhibition of metastasis overthe administration time in the non-treated group, in the group treatedwith the compound of Example 2, and in the positive control group,observed by image signal measurement.

FIG. 10 is a photograph illustrating the inhibition of metastasis in thelung tissue extracted from the nude mouse according to ExperimentalExample 8, observed by image signal measurement.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hereinafter, the present invention is described in detail.

The present invention provides the novel disubstituted adamantylderivative represented by the below formula 1 or the pharmaceuticallyacceptable salts thereof:

In the formula 1,

R¹ is —X—(CH₂)_(n)—R²;

R² is H or C1-C6 straight or branched alkyl;

R³ is unsubstituted or substituted C5-C10 aryl or unsubstituted orsubstituted heteroaryl,

wherein the said heteroaryl is 5-membered or 6-membered heteroarylcontaining one or more heteroatoms selected from the group consisting ofN, O, and S,

the said substituted aryl or heteroaryl can be substituted with one ormore halogens; C1-C6 straight or branched alkyl; hydroxy; C1-C6 straightor branched alkoxy; nitro; nitrile; unsubstituted amine or aminesubstituted with one or more C1-C6 straight or branched alkyls; C1-C6straight or branched alkylcarbonyl; C1-C6 straight or branchedalkoxycarbonyl or 5-membered or 6-membered heterocycloalkyl containingheteroatoms selected from the group consisting of N, O, and S;

X can be NH or O; at this time, when R³ is unsubstituted or substitutedaryl, X is O; and

n is an integer of 1-5.

Preferably,

R¹ is —X—(CH₂)_(n)—R³;

R² is H or C1-C4 straight or branched alkyl;

R³ is unsubstituted or substituted phenyl, unsubstituted or substitutedpyridine, pyrazine, imidazole, thiophene, benzothiophene, furan, orbenzofuran,

wherein the said substituted phenyl or the substituted pyridine,pyrazine, imidazole, thiophene, benzothiophene, furan or benzofuran canbe substituted with one or more fluoro, bromo, chloro, methyl, ethyl,propyl, isopropyl, butyl, hydroxy, t-butyl, methoxy, ethoxy, propoxy,butoxy, nitro, nitrile, amine, methylamine, dimethylamine, ethylamine,diethylamine, acetyl, ethylcarbonyl, methoxycarbonyl, ethoxycarbonyl, or5-membered or 6-membered heterocycloalkyl comprising heteroatomsselected from the group consisting of N, O, and S;

X can be NH or O; at this time, when R³ is unsubstituted or substitutedphenyl, X is O; and

n is an integer of 1-3.

More preferably,

R¹ is —X—(CH₂)_(n)—R³;

R² is methyl;

R³ is unsubstituted or substituted phenyl, or unsubstituted orsubstituted furan,

wherein the said substituted phenyl or the substituted heteroaryl can besubstituted with one or more chloro, bromo, methyl, ethyl, hydroxy,methoxy, ethoxy, ethylamine, acetyl, piperidine, piperazine, pyrolidine,tetrahydrofuran, or tetrahydrothiophene;

X can be NH or O; at this time, when R³ is unsubstituted or substitutedphenyl, X is O; and

n is an integer of 1-3.

Most preferably, the disubstituted adamantyl derivative represented byformula 1 is as follows:

(1)methyl-3-(2-(4-(3-((furan-2-ylmethoxy)carbonyl)adamantane-1-yl)phenoxy)acetamido)benzoate;or

(2)methyl-3-(2-(4-(4-((3,4-dimethoxybenzyloxy)carbonyl)adamantane-1-yl)phenoxy)acetamido)benzoate.

The disubstituted adamantyl derivative represented by formula 1 of thepresent invention can be used as the form of a pharmaceuticallyacceptable salt, in which the salt is preferably acid addition saltformed by pharmaceutically acceptable free acids. The acid addition saltcan be obtained from inorganic acids such as hydrochloric acid, nitricacid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid,nitrous acid and phosphorous acid, or non-toxic organic acids such asaliphatic mono/dicarboxylate, phenyl-substituted alkanoate, hydroxyalkanoate, alkandioate, aromatic acids and aliphatic/aromatic sulfonicacids. The pharmaceutically non-toxic salts are exemplified by sulfate,pyrosulfate, bisulfate, sulphite, bisulphite, nitrate, phosphate,monohydrogen phosphate, dihydrogen phosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, fluoride, acetate, propionate,decanoate, caprylate, acrylate, formate, isobutylate, caprate,heptanoate, propiolate, oxalate, malonate, succinate, suberate,cabacate, fumarate, maliate, butyne-1,4-dioate, hexane-1,6-dioate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, terephthalate,benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate,xylenesulfonate, phenylacetate, phenylpropionate, phenylbutylate,citrate, lactate, hydroxybutylate, glycolate, malate, tartrate,methanesulfonate, propanesulfonate, naphthalene-1-sulfonate,naphthalene-2-sulfonate and mandelate.

The acid addition salt in this invention can be prepared by theconventional method known to those in the art. For example, thedisubstituted adamantyl derivative represented by formula 1 is dissolvedin an organic solvent such as methanol, ethanol, acetone,methylenechloride, or acetonitrile, to which organic acid or inorganicacid is added to induce precipitation. Then, the precipitate is filteredand dried to give the salt. Or the solvent and the excessive acid aredistillated under reduced pressure, and dried to give the salt. Or theprecipitate is crystallized in the organic solvent to give the same.

A pharmaceutically acceptable metal salt can be prepared by using abase. Alkali metal or alkali earth metal salt is obtained by thefollowing processes: dissolving the compound in excessive alkali metalhydroxide or alkali earth metal hydroxide solution; filteringnon-soluble compound salt; evaporating the remaining solution, anddrying thereof. At this time, the metal salt is preferably prepared inthe pharmaceutically suitable form of sodium, potassium, or calciumsalt. And the corresponding silver salt is prepared by the reaction ofalkali metal or alkali earth metal salt with proper silver salt (ex;silver nitrate).

The present invention includes not only the disubstituted adamantylderivative represented by formula and the pharmaceutically acceptablesalts thereof but also solvates, hydrates, or isomers possibly producedfrom the same.

The present invention also provides a method for preparing thedisubstituted adamantyl derivative represented by formula 1 wherein thecompound represented by formula 2 is reacted with the compoundrepresented by formula 3 to give the compound represented by formula 1,as shown in the below reaction formula 1.

(In the reaction formula 1, R¹ and R² are as defined in formula 1).

The method for preparing the disubstituted adamantyl derivativerepresented by formula 1 of the present invention is to prepare thecompound represented by formula 1 via the coupling reaction betweencarboxyl group of the compound represented by formula 2 and the compoundrepresented by formula 3 in the presence of a base and a coupling agent.

At this time, as the coupling agent used in the above coupling reaction,diisopropylethylamine (DIPEA), triethylamine (TEA), ordimethylaminopyridine (DMAP) can be used along withbenzotriazole-1-yl-oxy-tris(dimethylamino)-phosphoniumhexafluorophosphate(Py-BOP),O-benzotriazole-N,N,N,N-tetramethyl-uronium-hexafluoro-phosphate (HBTU),2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate(HATU), hydroxybenzotriazole (HOBt), dicyclohexylcarbodiimide (DCC),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), orcarbonyldiimidazole (CDI), and more preferably DIPEA, TEA, or DMAP canbe used together with hydroxybenzotriazole (HOBt) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

The organic solvent used herein is exemplified by methanol,tetrahydrofuran (THF), dimethylformamide (DMF), dichloromethane (DCM),and toluene, which do not affect the reaction, and more preferablydimethylformamide (DMF) can be used.

Further, the present invention provides a pharmaceutical composition forthe inhibition of metastasis comprising the disubstituted adamantylderivative represented by formula 1 or the pharmaceutically acceptablesalts thereof as an active ingredient.

(In the formula 1, R¹ and R² are as defined in formula 1).

The following experiment was performed to investigate the inhibitoryeffect of the disubstituted adamantyl derivative represented by formula1 of the present invention on cancer cell metastasis by suppressing theaccumulation of HIF-1α. As a result, it was confirmed that thedisubstituted adamantyl derivative of the present invention suppressedaccumulation of HIF-1α leading inhibition of the expression of themetastasis related protein Twist, β-catenin, RohA and EMT related genessuch as MMP2 and MMP9.

Therefore, the anticancer activity to inhibit the cancer cell growth andmetastasis of the compound of the present invention was not attributedto the non-selective cytotoxicity but attributed to the selectiveinhibition of the expression of Twist (see Experimental Examples 1-8).

The disubstituted adamantyl derivative of the present invention couldinhibit the expression of the metastasis-related protein, Twist,dose-dependently by inhibiting metastasis related HIF-1, so that itdisplayed not only metastasis inhibiting effect by suppressing theexpression of metastasis-related proteins like β-catenin, Twist, andRohA and EMP related genes like MMP2 and MMP9 but also no side effectscaused by cytotoxicity, indicating that the compound could beefficiently used as a pharmaceutical anticancer composition or apharmaceutical composition for inhibiting metastasis.

In the pharmaceutical composition for inhibiting metastasis or thepharmaceutical anticancer composition of the present invention, the saidcancer is a solid cancer, which is exemplified by colorectal cancer,liver cancer, stomach cancer, breast cancer, colon cancer, bone cancer,pancreatic cancer, head & neck cancer, uterine cancer, ovarian cancer,rectal cancer, esophageal cancer, small bowel cancer, anal cancer, coloncancer, fallopian tube carcinoma, endometrial carcinoma, uterinecervical carcinoma, vaginal carcinoma, vulval carcinoma, Hodgkin'sdisease, prostate cancer, bladder cancer, kidney cancer, ureter cancer,renal cell carcinoma, renal pelvic carcinoma, and central nervous systemtumor.

The present invention also provides a pharmaceutical anticancercomposition comprising the disubstituted adamantyl derivativerepresented by formula 1 or the pharmaceutically acceptable saltsthereof as an active ingredient.

(In the formula 1, R¹ and R² are as defined in formula 1).

The following experiment was performed to investigate whether or not thedisubstituted adamantyl derivative represented by formula 1 of thepresent invention could inhibit the expression of Twist gene byinhibiting HIF-1α accumulation under hypoxia. As a result, the compoundof the present invention was confirmed to have the dose-dependentinhibitory effect on HRE activity, and thereby it could inhibit theexpression of Twist protein involved in metastasis and cancer cellproliferation. Therefore, it was confirmed that the anticancer activityto inhibit the cancer cell growth and metastasis of the compound of thepresent invention was not attributed to the non-selective cytotoxicitybut attributed to the selective inhibition of the expression of Twist(see Experimental Examples 1-8).

The disubstituted adamantyl derivative of the present inventioninhibited accumulation of HIF-1α to suppress the expression of Twistprotein dose-dependently, suggesting that the compound was not onlyexcellent in inhibiting metastasis and cancer cell proliferation butalso free from side effects caused by cytotoxicity when it was absorbedin a living body. Therefore, it could be efficiently used as apharmaceutical anticancer composition.

The pharmaceutical composition comprising the disubstituted adamantylderivative of formula 1 or the pharmaceutically acceptable salts thereofas an active ingredient of the present invention can be administeredorally or parenterally and be used in general forms of pharmaceuticalformulation, but not always limited thereto.

The formulations for oral administration are exemplified by tablets,pills, hard/soft capsules, solutions, suspensions, emulsions, syrups,granules, and elixirs, etc. These formulations can include diluents (forexample, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose,and/or glycine) and lubricants (for example, silica, talc, stearate andits magnesium or calcium salt, and/or polyethylene glycol) in additionto the active ingredient. Tablets can include binding agents such asmagnesium aluminum silicate, starch paste, gelatin, methylcellulose,sodium carboxymethylcellulose and/or polyvinylpyrolidone, and ifnecessary disintegrating agents such as starch, agarose, alginic acid orits sodium salt or azeotropic mixtures and/or absorbents, coloringagents, flavors, and sweeteners can be additionally included thereto.

The pharmaceutical composition comprising the disubstituted adamantylderivative of formula 1 as an active ingredient of the present inventioncan be administered by parenterally and the parenteral administrationincludes subcutaneous injection, intravenous injection, intramuscularinjection and intrathoracic injection.

To prepare the composition as a formulation for parenteraladministration, the disubstituted adamantyl derivative represented byformula 1 or the pharmaceutically acceptable salts thereof of thepresent invention are mixed with a stabilizer or a buffering agent toproduce a solution or suspension, which is then formulated as ampoulesor vials. The composition herein can be sterilized and additionallycontains preservatives, stabilizers, wettable powders or emulsifiers,salts and/or buffers for the regulation of osmotic pressure, and othertherapeutically useful materials, and the composition can be formulatedby the conventional mixing, granulating or coating method. The effectivedosage of the pharmaceutical composition comprising the disubstitutedadamantyl derivative represented by formula 1 as an active ingredient ofthe present invention is 0.01-200 mg/kg per day, which can beadministered orally or parenterally several times a day or preferablyonce a day or three times a day.

The pharmaceutical composition of the present invention can beadministered alone or together with surgical operation, hormone therapy,chemo-therapy and biological regulators.

Practical and presently preferred embodiments of the present inventionare illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, onconsideration of this disclosure, may make modifications andimprovements within the spirit and scope of the present invention.

1. Analysis Devices

The analysis devices used in this invention to investigate the structureof the product of the invention were as follows.

For nuclear magnetic resonance (¹H NMR), Varian 300

MHz spectrometer and Varian 400 MHz spectrometer were used. CDCl₂,MeOH-d₄, and DMSO-d₆ were used as NMR solvents.

2. TLC and Column Chromatography

Thin layer chromatography (TLC) was performed with 0.25 mm silica plate(Merck F254), the product of E. Merck Co. Silica gel used for columnchromatography was Merck Em9385, 230-400 mesh. To investigate thematerial separated on TLC, UV lamp (=254 nm) was used or the materialwas exposed on iodine vapor. Or the material was soaked in PMA,ninhydrin solution, p-anisaldehyde, or KMnO₄ to induce colordevelopment, followed by heating.

3. Reagents

The reagents used in this invention were purchased from Sigma-Aldrich,Lancaster, Fluka, and TC1. Tetrahydrofuran (THF) used for the reactionwas prepared by the reaction between Na metal and benzophenone in argonenvironment via heat-refluxing. When THF turned blue, it was used.Dichloromethane (CH₂Cl₂) was reacted with CaH₂ in argon environment viaheat-refluxing. Other solvents used herein were the 1^(st) degreereagents purchased from Sigma-Aldrich, which were used without beingpurified. Ethylacetate and n-hexane were purified by heat-refluxing inargon environment before being used.

Manufacturing Example 1 Preparation of 3-bromo-adamantane-1-carboxylicacid

A two-neck round bottom flask was connected to a reflux condenser, towhich aluminum chloride (AlCl₃, 7.15 mmol) was injected, followed bycooling at −5° C. in argon environment. Then, bromine (Br₂, 66 mmol) wasadded thereto, followed by stirring for 15 minutes.1-adamantanecarboxylic acid (5.5 mmol) was added to the reaction mixtureat a time, followed by stirring for 1 hour with maintaining thetemperature at −5° C. The reaction mixture was then additionally stirredfor 48 hours with raising the temperature slowly to room temperature.Upon completion of stirring, ice water was poured to the mixture toterminate the reaction. Excessive bromine was de-colored by addingsodium pyrosulfite. The de-colored reactant was extracted by usingchloroform. The extracted organic layer was dried over sodium sulfate,followed by concentration under reduced pressure to give the targetcompound (5.016 mmol, 92.83%).

¹H NMR (300 MHz, CDCl₃): δ 2.49 (2H, s), 2.36-2.22 (6H, m), 1.9 (2H, m),1.71 (2H, s).

Manufacturing Example 2 Preparation of3-(4-methoxyphenyl)adamantane-1-carboxylic acid

Aluminum chloride (AlCl₃, 7.7 mmol) was dissolved in anisol (96.25mmol), which was cooled down at −10° C. The compound prepared inManufacturing Example 1 (3.85 mmol) was added thereto, followed bystirring for 24 hours with raising the temperature slowly to roomtemperature. Upon completion of stirring, ice water in whichhydrochloric acid (3.3 ml) was dissolved was poured to the reactionmixture to terminate the reaction, followed by extraction withethylacetate (EA). The extract was then dried over sodium sulfate. Thedried reactant was concentrated under reduced pressure, followed bysolidification with hexane. The obtained solid was filtered to give thetarget compound (2.968 mmol, 77%).

¹H NMR (300 MHz, CDCl₃): δ 7.29 (2H, d, J=8.7 Hz), 6.87 (2H, d, J=8.7Hz), 3.79 (3H, s), 2.23-1.73 (14H, m).

Manufacturing Example 3 Preparation of3-(4-hydroxyphenyl)adamantane-1-carboxylic acid

The compound (3.5 mmol) prepared in Manufacturing Example 2 wasdissolved in dichloromethane (DCM), followed by cooling at −10° C., towhich borontribromide (BBr₃, 1.0 M dichloromethane solution, 2.5equivalent) was loaded in argon environment. Upon completion of theloading, the temperature was raised to room temperature while stirringfor 3 hours. Then, cold water was poured to the reaction mixture toterminate the reaction. The reactant was extracted with ethylacetate,and then the extract was dried over sodium sulfate. The dried extractwas concentrated under reduced pressure, and purified by columnchromatography to give the target compound (2.57 mmol, 73.6%).

¹H NMR (300 MHz, DMSO-d₆): δ 12.00 (1H, bs), 9.12 (1H, bs), 7.14 (2H, d,J=8.7 Hz), 6.69 (2H, d, J=8.7 Hz), 2.12-1.65 (14H, m).

Manufacturing Example 4 Preparation of3-(4-hydroxyphenyl)adamantane-1-carboxylic acid

The compound (2.46 mmol) prepared in Manufacturing Example 3 wasdissolved in dimethylformamide (3.5 ml), to which anhydrouspotassiumbicarbonate (KHCO₃, 2.95 mmol) was added, followed by stirringat room temperature for 15 minutes. Benzylbromide (3.7 mmol) was addedthereto, followed by stirring for 4 more hours with raising thetemperature to 40° C. Upon completion of the stirring, the reactionmixture was diluted in saturated sodiumbicarbonate solution, followed byextraction with ethylacetate (EA). The extract was dried over sodiumsulfate. The dried reactant was purified by column chromatography togive the target compound (154 mmol, 62.6%).

¹H NMR (300 MHz, DMSO-d₆): δ 9.12 (1H, bs), 7.36 (5H, m), 7.14 (2H, d,J=8.7 Hz), 6.69 (2H, d, J=8.7 Hz), 5.08 (2H, s), 2.14-1.66 (14H, m).

Manufacturing Example 5 Preparation of3-(4-ethoxycarbonylmethoxyphenyl)adamantane-1-carboxylic acidbenzylester

The compound (0.55 mmol) prepared in Manufacturing Example 4,potassiumcarbonate (0.66 mmol), and ethylchloroacetate (1.65 mmol) weredissolved in dimethylformamide (DMF, 1 ml), followed by stirring at roomtemperature for overnight. Upon completion of the reaction, the reactionmixture was diluted in ethylacetate (EA) and then washed with saturatedsodiumbicarbonate and brine, followed by drying over sodium sulfate. Thedried reactant was purified by column chromatography to give the targetcompound (0.533 mmol, 96.7%).

¹H NMR (300 MHz, CDCl₃): δ 7.35 (5H, m), 7.28 (2H, d, J=8.7 Hz), 6.87(2H, d, J=8.7 Hz), 5.11 (2H, s), 4.59 (2H, s), 4.30 (2H, q, J=7.5 Hz),2.22-1.71 (14H, m), 1.32 (3H, t, J=7.2 Hz).

Manufacturing Example 6 Preparation of3-(4-carboxymethoxyphenyl)adamantane-1-carboxylic acid benzylester

The compound prepared in Manufacturing Example 5 (0.512 mmol) wasdissolved in the mixed solution of tetrahydrofuran/distilled water (1:1)(2.5 ml), to which lithiumhydroxide monohydrate (LiOH H₂O, 1.024 mmol),followed by stirring at room temperature for 2 hours. Upon completion ofstirring, the reaction mixture was neutralized with 10% hydrochloricacid, followed by extraction with ethylacetate (EA). The extractedorganic layer was dried over sodium sulfate, followed by concentrationunder reduced pressure. The concentrated reactant was purified by columnchromatography to give the target compound (0.33 mmol, 65%).

¹H NMR (300 MHz, CD₃OD): δ 7.34 (5H, m), 7.28 (2H, d, J=8.7 Hz), 6.88(2H, d, J=8.7 Hz), 5.10 (2H, s), 4.58 (2H, s), 4.30 (2H, q, J=7.5 Hz),2.19-1.75 (14H, m).

Manufacturing Example 7 Preparation of3-(4-[(3-methoxycarbonylphenylcarbamoyl)methoxy]phenyl}adamantane-1-carboxylicacid benzylester

The compound (0.33 mmol) prepared in Manufacturing Example 6,3-aminobenzoic acid methylester (0.66 mmol),benzotriazole-1-yl-oxy-tris(dimethylamino)-phosphoniumhexafluorophosphate(Py-BOP, 0.66 mmol), and dimethylaminopyridine (DMAP, 0.66 mmol) weredissolved in dimethylformamide (DMF, 1 ml), followed by stirring at roomtemperature for overnight. Upon completion of stirring, the reactionmixture was diluted in ethylacetate (EA) and then washed with 10%hydrochloric acid, brine, and water, followed by drying over sodiumsulfate. The dried reactant was purified by column chromatography togive the target compound (0.25 mmol, 75.3%).

¹H NMR (300 MHz, CDCl₃): δ 8.38 (1H, s), 8.07 (1H, s), 8.01 (1H, d),7.83 (1H, d), 7.45 (1H, t, J=8.0 Hz), 7.31-7.38 (7H, m), 6.96 (2H, d),5.12 (2H, s), 4.61 (2H, s), 3.93 (3H, s), 2.24 (2H, s), 2.04 (2H, s),1.92 (5H, t, J=15.2 Hz), 1.88 (4H, s), 1.74 (2H, s).

Manufacturing Example 8 Preparation of3-(4-[(3-carboxyphenylcarbamoyl)methoxy]phenyl}adamantane-1-carboxylicacid benzylester

The compound prepared in Manufacturing Example 7 was dissolved in themixed solution of tetrahydrofuran/distilled water/dioxane=1:1:1 (2.5ml), to which lithiumhydroxide monohydrate (LiOH H₂O, 0.18 mmol) wasadded, followed by stirring at room temperature for 4 hours. Uponcompletion of stirring, the reaction mixture was diluted in ethylacetate(EA) and then neutralized with 10% hydrochloric acid, followed bywashing with brine. The organic layer was extracted from the washedreactant, which was dried over sodium sulfate and concentrated underreduced pressure. The concentrated reactant was purified by columnchromatography to give the target compound (0.072 mmol, 80.03%).

¹H NMR (300 MHz, DMSO-d₆): δ 13.00 (1H, bs), 10.24 (1H, s), 8.27 (1H,s), 7.88 (1H, d, J=8.7 Hz), 7.66 (1H, d, J=8.1 Hz), 7.46 (1H, t, J=8.1Hz), 7.37 (7H, m), 6.95 (2H, d, J=8.1 Hz), 5.09 (2H, s), 4.67 (2H, s),2.16-1.67 (14H, m).

Example 1 Preparation ofmethyl-3-(2-(4-(3-((furan-2-ylmethoxy)carbonyl)adamantane-1-yl)phenoxy)acetamido)benzoate

The compound (60 mg, 0.13 mmol) prepared in Manufacturing Example 8 wasdissolved in tetrahydrofuran, which was cooled down at −10° C.2-(bromomethyl)furan (0.02 ml, 0.17 mmol), ethylchloroformate (0.02 ml,15 mmol), and triethylamine (0.02 ml, 0.15 mmol) were loaded thereto.After stirring the reaction mixture at room temperature for 30 minutes,water was poured in to terminate the reaction. The reactant wasextracted with ethylacetate (EA), and the extracted organic layer wasdried over magnesium sulfate, followed by concentration under reducedpressure. The concentrated reactant was purified by columnchromatography (silica, dichloromethane/methanol) to give the targetcompound (49 mg, 70%).

¹H NMR (400 MHz, CDCl₃): δ 8.37 (1H, s), 8.07 (1H, s), 8.00-8.02 (1H,m), 7.83 (1H, d), 7.45 (1H, t, J=8.4 Hz), 6.96 (2H, d), 6.34 (3H, d),6.31-6.33 (1H, m), 6.21 (1H, d), 5.89 (1H, s), 4.61 (2H, s), 4.44 (2H,d), 3.93 (3H, s), 1.98 (2H, s), 1.89-1.90 (8H, m), 1.74 (2H, s).

Example 2 Preparation ofmethyl-3-(2-(4-(4-((3,4-dimethoxybenzyloxy)carbonyl)adamantane-1-yl)phenoxy)acetamido)benzoate

The compound (307.9 mg, 0.66 mmol) prepared in Manufacturing Example 8,3,4-dimethoxybenzylalcohol (0.14 ml, 0.99 mmol),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (189.8 mg,0.99 mmol), 1-hydroxybenzotriazole hydrate (133.8 mg, 0.99 mmol), andN,N-diisopropylethylamine (0.17 ml, 0.99 mmol) were dissolved indimethylformamide at room temperature, followed by stirring forovernight. Upon completion of stirring, water was added thereto toterminate the reaction, followed by extraction with ethylacetate (EA).The extracted organic layer was dried over anhydrous magnesium sulfate,followed by concentration under reduced pressure. The concentratedreactant was purified by column chromatography (silica gel,dichloromethane/ethylacetate) to give the target compound (368.6 mg,91%).

¹H NMR (400 MHz, CDCl₃): δ 8.38 (1H, s), 8.08 (1H, s), 8.01 (1H, d),7.83 (1H, d), 7.45 (1H, t, J=8.0 Hz), 6.90-6.94 (3H, m), 6.85 (2H, d),5.06 (1H, s), 4.61 (1H, s), 3.93 (3H, s), 3.88 (1H, s), 2.20 (1H, s),1.98 (1H, s), 1.94 (1H, s), 1.88 (81, s), 1.73 (1H, s).

Experimental Example 1 Inhibition of Transcriptional Activation Mediatedby HIF-1

The following experiment was performed to investigate the antagonism ofthe disubstituted adamantyl derivative of the invention withaccumulation of HIF-1α.

The compound that can inhibit HRE transcriptional activity mediated byHIF-1α under hypoxia is also able to inhibit metastasis and cancer cellproliferation. Thus, the present inventors first investigated theinhibition of HRE transcriptional activity to confirm HIF-1αsuppression. Particularly, to determine the suppression of HREtranscriptional activity by the compounds prepared in Example 1 andExample 2, HRE (Hypoxia Responsive Element, 5′-ACGTG-3′) located inhuman VEGFA gene was duplicated 6 copies in the multi-cloning site ofpGL3-basic vector (Promega) using luciferase as a reporter, resulting inthe construction of pGL3-HRE-luciferase.

The human colorectal carcinoma cell line HCT116 (ATCC #: CCL-247) wasseeded in a 48-well cell culture plate. On the next day, the cells weretransfected with ng of pGL3-HRE-luciferase vector and 2.5 ng of Renilla,the control vector, by using polyfect reagent. After 24 hours ofculture, the medium was replaced, followed by further culture for 4hours. The cells were treated with the compounds prepared in Example 1and Example 2 of the invention at the concentrations of 0, 0.3125,0.623, 1.25, 2.5, 5, and 10 μM, followed by culture for 12 hours underhypoxia (O₂ 1%, N₂ 94%, CO₂ 5%). Cell lysate was obtained by using RIPAbuffer, followed by the measurement of the luciferase activity inducedunder hypoxia by dual-luciferase assay (Promega), measuring HREinhibition activity of the compound of Example 2. The results are shownin FIG. 1.

As shown in FIG. 1, the compounds prepared in Examples of the presentinvention were able to inhibit HRE transcription in dose-dependentmanner. In particular, IC₅₀ of the compound prepared in Example 2 was0.86 μM, indicating significant HRE transcription inhibition effect.Therefore, the compounds of the present invention were confirmed to beexcellent in inhibition of HRE transcription by HIF-1α induced underhypoxia.

The adamantyl derivative of the present invention is excellent insuppression of HIF-1α related to metastasis and proliferation of cancercells, suggesting it can be efficiently used as a pharmaceuticalanticancer composition or a pharmaceutical composition inhibitingmetastasis.

Experimental Example 2 Inhibition of HIF-1α Accumulation Under Hypoxia

The following experiment was performed to investigate the inhibition ofHIF-1α accumulation induced by the disubstituted adamantyl derivative ofthe invention under hypoxia.

In this experiment, it was investigated whether or not the accumulationof HIF-1α could be inhibited by the compound prepared in Example 2demonstrating excellent inhibition effect of HRE transcription in theHCT116 and HT1080. First, HCT116 (ATCC #: CCL-247) and HT1080 (ATCC#CCL-121) were seeded in cell culture vessels at the density of 2×10⁵cell/ml, followed by culture for 24 hours. The cells were thenpre-treated under hypoxia (O₂ 1%, N₂ 94%, CO₂ 5%) for 4 hours to inducethe accumulation of HIF-1α. Then, the cells were treated with thecompound prepared in Example 2 at the concentrations of 0, 1, 3, and 10μM, followed by culture for 12 hours under hypoxia. Cell extract wasprepared by using RIPA buffer. At this time, to compare the expressionof HIF-1 target gene under hypoxia, the control having oxygen 20% wasprepared. The nuclear extract 30 μg was separated from each sample bySDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis),which was transferred onto polyvinylidene fluoride membrane. Then,HIF-1α protein therein was measured by using HIF-1α antibody (R&DSystem) and HRP (horseradish peroxidase) labeled secondary antibody(Amersham-Pharmacia). GAPDH (glyceraldehyde 3-phosphate dehydrogenase)was used as the control protein. The results are shown in FIG. 2.

As shown in FIG. 2, the compound prepared in Example 2 did not affectthe generation of GAPDH, while the compound did inhibit the accumulationof HIF-1α induced under hypoxia in a dose-dependent manner. From theabove results, it was confirmed that the compound of the presentinvention can inhibit the expression of HIF-1α by suppressing theaccumulation of HIF-1α induced under hypoxia in cancer cells.

Since the disubstituted adamantyl derivative of the present invention issignificant in inhibiting the accumulation of HIF-1α that is involved inmetastasis and proliferation of cancer cells, the compound can beefficiently used as a pharmaceutical anticancer or antimetastasiscomposition.

Experimental Example 3 Inhibitory Effect of the Compound of theInvention on the Expression of Metastasis Related Gene

The following experiment was performed to investigate whether or not thedisubstituted adamantyl derivative of the present invention inhibitedthe expression of metastasis related gene by suppressing the expressionof HIF-1α.

First, the human colorectal carcinoma cell line HCT116 (ATCC #: CCL-247)was seeded in cell culture vessels at the density of 2×10⁵ cell/ml,followed by culture for 24 hours. The cells were then pre-treated underhypoxia (O₂ 1%, N₂ 94%, CO₂ 5%) for 4 hours to induce the accumulationof HIF-1α. Then, the cells were treated with the compound of theinvention at the concentrations of 0, 1, 3, and 10 μM, followed byculture for 12 hours under hypoxia. RNA was purified using trizol. Tocompare the expression of HIF-1 target gene under hypoxia, the controlcontaining oxygen 20% was prepared. The amount of mRNAs of EMT relatedgenes such as MMP2 and MMP9 and uPA, metastasis related genes induced byHIF-1α, were measured by RT-PCR kit (Invitrogen). At this time, GAPDH,as an internal gene, was simultaneously amplified to investigate theselective inhibition activity of the compound prepared in Example 2against MMP2, MMP9, and uPA. The results are shown in FIG. 3.

As shown in FIG. 3, the compound prepared in Example 2 inhibitedexpression of MMP2, MMP9, and uPA by inhibiting accumulation of HIF-1αunder hypoxia. In particular, the expressions of MMP2 and MMP9 weresignificantly inhibited by the compound prepared in Example 2 in adose-dependent manner.

Therefore, the disubstituted adamantyl derivative of the presentinvention can be efficiently used as a pharmaceutical composition ofdrugs for anticancer or antimetastasis, since it can inhibit expressionof EMT proteins stimulating metastasis by suppressing HIF-1α.

Experimental Example 4 Inhibitory Effect of the compound of theInvention on the Induction of EMT

The inhibitory effect of the present invention was evaluated on theinduction of EMT using EMT inducing proteins such as β-catenin, RohA,vimentin, and Twist.

First, the human colorectal carcinoma cell line HCT116 (ATCC #: CCL-247)was seeded in cell culture vessels at the density of 2×10⁵ cell/ml,followed by culture for 24 hours. The cells were then pre-treated underhypoxia (O₂ 1%, N₂ 94%, CO₂ 5%) for 4 hours to induce the accumulationof HIF-1α. Then, the cells were treated with the compound of theinvention at the concentrations of 0, 1, 3, and 5 μM, followed byculture for 12 hours under hypoxia. Cell extract was prepared by usingRIPA buffer. At this time, to compare the expression of HIF-1 targetgene under hypoxia, the control having oxygen 20% was prepared. Thenuclear extract 30 μg was separated from each sample by SDS-PAGE (sodiumdodecyl sulfate-polyacrylamide gel electrophoresis), which wastransferred onto polyvinylidene fluoride membrane. Then, the amount ofβ-catenin, RhoA, vimentin, and Twist was measured by using the primaryantibody against each of β-catenin, RhoA, vimentin, and Twist, and HRP(horseradish peroxidase) labeled secondary antibody(Amersham-Pharmacia). GAPDH (glyceraldehyde 3-phosphate dehydrogenase)was used as the control protein. The results are shown in FIG. 4.

As shown in FIG. 4, the compound prepared in Example 2 inhibited themetastasis related protein Twist. The compound also inhibited theexpression of β-catenin and RhoA that regulates cancer cell metastasis.The inhibition of the expression of the metastasis related protein bythe compound prepared in Example 2 was not attributed to itsnon-selective cytotoxicity but attributed to the selective inhibition ofthe expression of Twist.

Therefore, the disubstituted adamantyl derivative of the presentinvention can be efficiently used as a pharmaceutical anticancer orantimetastasis composition, since it can inhibit the activity of Twistto inhibit the expression of the metastasis related proteins bysuppressing accumulation of HIF-1α.

Experimental Example 5 Inhibitory Effect of the Compound of theInvention on Cell Migration

The following experiment was performed to evaluate the inhibitory effectof the compound prepared in Example 2 on cell migration.

First, an insert (Ibidi) was placed in the center of a 6-well cultureplate by using a pincette. 70 μl of the human colorectal carcinoma cellline HCT116 was loaded in each well at the density of 3×10⁵ cell/ml.Medium was filled around the insert. 24 hours later, the insert wascarefully removed by using a pincette. The cells were carefully washedwith medium not to be fallen off to the bottom. Then, the diluted mediumsupplemented with the compound prepared in Example 2 (0.4 μM) was addedthereto. 48 hours later, cell migration was observed under microscope.At this time, the medium containing dimethylsulfoxide (DMSO) was usedfor the non-treated group. Cell migration was photographed by usingreal-time cell analyzer (Applied Biophysics). First, 100 μl of cysteinesolution was loaded in a 8-well plate, which stood at room temperaturefor 10 minutes. 2×10⁵ HCT116 cells were suspended in 300 μl of medium.Cysteine solution was eliminated and then the cells were added, followedby culture in a 37° C. incubator for 15 hours. When saturation of thecell was confirmed on the graph of real-time cell analyzer, a roundwound was made by electric shock. Then, the medium was replaced with themedium supplemented with the compound prepared in Example 2 (10 μM). Thecell growth around the round wound was observed. Time that had taken forthe round wound to be filled up with new grown cells was measured andcompared with that of the control, which was calculated on the graph.The results are shown in FIG. 5 and FIG. 6.

As shown in FIG. 5 and FIG. 6, in the cancer cells treated with thecompound of the invention, cell migration was inhibited so that theround wound remained unfilled. In the meantime, in the non-treatedgroup, cell migration was confirmed and the round wound was filled up.From the graph obtained by real-time cell analyzer, it was confirmedthat time for cell migration in the non-treated group was 11.5 hours,which was comparatively short, while the time for cell migration in thecells treated with the compound prepared in Example 2 of the inventionwas 22 hours, which was almost double the time of the non-treated group,indicating at least double the cell migration inhibiting effect.

The disubstituted adamantyl derivative of the present inventioninhibited cancer cell migration by suppressing the expression of HIF-1α.The cancer cell migration inhibiting effect of the disubstitutedadamantyl derivative of the invention was almost twice as high as thatof the control group not-treated with the compound, so that the compoundof the invention can be efficiently used as a pharmaceutical anticanceror antimetastasis composition.

Experimental Example 6 Inhibitory Effect of the Compound of theInvention on Cell Invasion

The following experiment was performed to evaluate the inhibitory effectof the disubstituted adamantyl derivative of the present invention oncell invasion.

A cell culture insert (BD Falcon, 8-m pore size) was fixed on a 24-wellplate, to which 100 μl of matrigel diluted in 20 fold was added, whichstood at 37° C. for at least 1 hour. 700 μl of medium supplemented with10% FBS was loaded in each well of the 24-well plate under the insert.HT1080 cells (1×10⁵) displaying high metastatic activity were suspendedin 300 μl of serum-free medium. The cells were treated with the compoundof the invention at the concentrations of 0, 10, and 20 μM. The 300cells treated with the compound of the invention were placed in theinsert, followed by culture in a 37° C. incubator for hours. The insertwas taken out and fixed in 10% formalin solution. The cells remaining inthe inside of the insert were eliminated by using cotton swab, followedby washing with water. The cells were stained with sulforhodamine B for1 hour, and then washed with acetic acid, which were then dried. Thecells were photographed under optical microscope and cell invasion wasobserved. Also, a membrane was sliced and dissolved in 10 mM Trissolution, followed by measuring OD₅₄₀. The results are shown in FIG. 7.

As shown in FIG. 7, invasion of HT1080 cells treated with the compoundprepared in Example 2 was not observed and cell morphology was stillmaintained. Therefore, it was confirmed that the compound of the presentinvention inhibited metastasis by suppressing the cell invasion ofHT1080 cells having high metastatic activity.

Therefore, the disubstituted adamantyl derivative of the presentinvention can be efficiently used as a pharmaceutical anticancer orantimetastasis composition, since it is excellent in inhibiting cellinvasion and accordingly excellent in inhibiting metastasis.

Experimental Example 7 In Vivo Inhibition of Cancer Cell Proliferationin the Mouse by Intraperitoneal Administration of the Compound of theInvention

The following experiment was performed to evaluate the inhibitory effectof the disubstituted adamantyl derivative of the present invention oncancer cell proliferation via intraperitoneal administration.

First, female Balb/c nude mice (6 weeks old) were administered with thecompound prepared in Example 2 via intraperitoneal injection. Aftergenerating a tumor in the nude mouse by transplanting the humancolorectal carcinoma cell line HCT116, the compound prepared in Example2 was intraperitoneally administered at the concentration of 30 mg/kgfor 1 week. One week later, any change in the tumor size was observed bycomparing the volume of the first formed tumor and the volume measuredone week after. On the last day, the tumor was weighed. To investigatetoxicity of the compound, any general symptoms along with the weightchanges in the animal were observed during the administration period. Atthis time, an excipient was treated to the non-treated group, andsunitinib (30 mg/kg) was administered to the positive control. All thevalues of each measurement title were examined by t-test to investigatethe statistical significance of the values obtained from the non-treatedgroup, the group treated with the compound of Example 2, and thepositive control group. The results are shown in Table 1, Table 2, andFIG. 8.

TABLE 1 Tumor weight at final day (mg) (Inhibition Tumor volume over therate of administration time (V_(t) − V_(o)) tumor (Inhibition rate oftumor growth growth (%)) (%)) 0 day 2 days 4 days 4 days 7 days Non- 0.0± 0.0 22.8 ± 2.5  72.2 ± 7.3 334.9 ± 44.6  1689.6 ± 227.3  treated groupCompound 0.0 ± 0.0 18.5 ± 2.5*  57.5 ± 6.7** 262.6 ± 38.5* 1318.7 ±220.9* of (0%) (18.9%) (20.3%) (21.6%) (22.0%) Example 2 Positive 0.0 ±0.0 15.6 ± 4.3*   47.5 ± 10.7**  205.4 ± 35.2***  1064.9 ± 130.9***control (0%) (31.7%) (34.2%) (38.7%) (37.0%) (sunitinib) 1 significant(t-Test): *p < 0.05, **p < 0.01, ***p < 0.001 (compare to non-treatedgroup)

TABLE 2 Weight changes over the administration time (%) 0 day 2 days 4days 7 days Non-treated 100.0 ± 0.0 101.7 ± 0.8 102.2 ± 01.8 102.3 ± 3.0group Compound of 100.0 ± 0.0 100.4 ± 1.3 100.8 ± 1.2   99.9 ± 1.3Example 2 Positive 100.0 ± 0.0 100.5 ± 1.4 100.7 ± 2.0  100.8 ± 3.3control (sunitinib)

As shown in Table 1, Table 2, and FIG. 8, when the compound prepared inExample 2 of the invention was administered alone, the inhibitory effecton tumor growth was 22%. When sunitinib was administered in the positivecontrol, the inhibitory effect on tumor growth was 37%. During theadministration of the compound of Example 2, none of specific symptomswere observed in the nude mice. The weights of the mice were not muchchanged or not much different from that of the non-treated group evenafter 7 days from the administration. Therefore, it was confirmed thatthe compound of the present invention had the inhibiting activity ofcancer cell proliferation without cytotoxicity.

The disubstituted adamantyl derivative of the present invention wasexcellent in inhibiting cancer cell proliferation without cytotoxicity,indicating that it is safe in human to be used as a pharmaceuticalanticancer or antimetastasis composition.

Experimental Example 8 Evaluation of Inhibition of Metastasis In Vivo inthe Test Mouse

The following experiment was performed to investigate the inhibition ofmetastasis by the disubstituted adamantyl derivative of the presentinvention in the test animal administered with the compound viaintraperitoneal injection.

To evaluate the inhibition effect of the compound on metastasis, C57BL/6mice were transplanted with B16F10 melanoma cells (2×10⁶ cells/ml)expressing luciferase via intravenous injection (0.2 ml, 4×10⁵cells/mouse). One hour later, 50 μl of luciferin was administered toeach mouse at the concentration of 15 mg/ml via intraperitonealinjection. Then, the compound prepared in Example 2 of the invention wasintraperitoneally administered at the concentration of 50 mg/kg 13times. At this time, dimethylsulfoxide (DMSO) was treated to thenon-treated group, and sunitinib (30 mg/kg), the conventional anticanceragent, was administered to the positive control. The image of the mousewas photographed every day for 14 days with a live animal imaging system(PHOTON IMAGER, Biospace). On the last day of the experiment, the mousewas sacrificed by using CO₂ gas. The lung was extracted from the mouse,and lung metastasis of the skin cancer cells was confirmed. Toinvestigate toxicity, weight and other general symptoms were observedduring the experimental period. At this time, all the values of eachmeasurement title were examined by t-test for the statisticalsignificance of the values obtained from the non-treated group, thegroup treated with the compound of Example 2, and the positive controlgroup. The results are shown in Table 3, Table 4, FIG. 9, and FIG. 10.

TABLE 3 Image signal at final day (Inhibition Image signals over therate of administration time (photons/s/sr) tumor (Inhibition rate oftumor metastasis metastasis (%)) (%)) 0 day 4 days 9 days 14 days 14days Non- 557.2 ± 407.2 164.3 ± 66.1  666.5 ± 1080.4 32145.5 ± 31121.381962.6 ± 53648.4 treated group Compound 1112.8 ± 1015.6 157.8 ± 23.2392.3 ± 534.8  4740.0 ± 1920.5*  14751.2 ± 9987.8** of (3.9%) (41.1%)(85.3%) (82.0%) Example 2 Positive 857.2 ± 679.3  222.0 ± 146.1 47.5 ±10.7 13084.0 ± 8931.2  21738.7 ± 14939.0 control (19.9%) (59.3%) (73.4%)(sunitinib) significant (t-Test): *p < 0.05, **p < 0.01 (compare tonon-treated group)

TABLE 4 Weight changes over the administration time (%) 0 day 2 days 4days 7 days 9 days 11 days 14 days Non-treated 100.0 ± 0.0 101.1 ± 4.7102.7 ± 3.0 104.5 ± 4.7 104.2 ± 6.0 107.3 ± 6.2 108.0 ± 3.9 groupCompound of 100.0 ± 0.0 102.7 ± 3.8 103.6 ± 2.8 103.9 ± 2.6 106.4 ± 3.6111.1 ± 4.7 111.2 ± 7.6 Example 2 Positive control 100.0 ± 0.0 100.9 ±2.2 102.9 ± 2.0 103.0 ± 2.8 106.0 ± 3.1 105.4 ± 2.9 107.7 ± 3.3(sunitinib)

As shown in Table 3, Table 4, FIG. 9, and FIG. 10, the experimentalgroup treated with the compound prepared in Example 2 displayed themetastasis inhibiting effect by 85.3% (p<0.05), compared with that ofthe non-treated treated group according to the signal (photons/s/sr). Inthe meantime, the positive control displayed the inhibition ofmetastasis by 59.3%. On the last day, the lung was extracted from themouse, followed by imaging. In the experimental group treated with thecompound of Example 2, metastasis was inhibited by 82.0% (P<0.01). Inthe positive control group, metastasis was inhibited by 73.4% (P<0.05).Specific symptoms were not observed in the mouse treated with thecompound of Example 2 during the whole period of experiment. The mousebody weight was not much changed or reduced, either, compared with thatof the non-treated group until the last day (day 14). Therefore, it canbe concluded that the inhibition of metastasis by the compound of thepresent invention is more significant than the conventional anticanceragent, sunitinib. Further, the compound of the invention is confirmed tobe safe without cytotoxicity.

The disubstituted adamantyl derivative of the present inventioninhibited accumulation of HIF-1α, thereby inhibited the expression ofthe metastasis related protein Twist, leading inhibition of themetastasis related proteins, β-catenin, and RohA, and EMT related genessuch as MMP2 and MMP9, suggesting excellent inhibition effect of theinvented compound on cancer cell metastasis. In addition, the compoundof the invention displays no cytotoxicity mediated side effects when itis absorbed in a living body. Therefore, it can be efficiently used as apharmaceutical anticancer or antimetastasis composition.

The disubstituted adamantyl derivative represented by formula 1 of thepresent invention can be formulated in various forms according to thepurpose of use. The followings are some examples of differentformulations of the composition comprising the compound represented byformula 1 as an active ingredient, but the present invention is notlimited thereto.

Manufacturing Example 1 Preparation of Powders

Compound of formula 1 2 g Lactose 1 g

Powders were prepared by mixing all the above components, which werefilled in airtight packs according to the conventional method forpreparing powders.

Manufacturing Example 2 Preparation of Tablets

Compound of formula 1 100 mg Corn starch 100 mg Lactose 100 mg Magnesiumstearate  2 mg

Tablets were prepared by mixing all the above components by theconventional method for preparing tablets.

Manufacturing Example 2 Preparation of Capsules

Compound of formula 1 100 mg Corn starch 100 mg Lactose 100 mg Magnesiumstearate  2 mg

Capsules were prepared by mixing all the above components, which werefilled in gelatin capsules according to the conventional method forpreparing capsules.

Manufacturing Example 4 Preparation of Injections

Compound of formula 1 100 mg Mannitol 180 mg Na₂HPO₄•2H₂O  26 mgDistilled water 2974 mg 

Injections were prepared by mixing all the above components by theconventional method for preparing injections.

Those skilled in the art will appreciate that the conceptions andspecific embodiments disclosed in the foregoing description may bereadily utilized as a basis for modifying or designing other embodimentsfor carrying out the same purposes of the present invention. Thoseskilled in the art will also appreciate that such equivalent embodimentsdo not depart from the spirit and scope of the invention as set forth inthe appended claims.

1. A disubstituted adamantyl derivative represented by formula 1 or apharmaceutically acceptable salt thereof:

wherein R¹ is —X—(CH₂)_(n)—R³; R² is H or a C1-C6 straight or branchedalkyl; and R³ is unsubstituted or substituted C5-C10 aryl orunsubstituted or substituted heteroaryl, wherein the heteroaryl is5-membered or 6-membered heteroaryl comprising one or more heteroatomsselected from the group consisting of N, O, and S, the substituted arylor heteroaryl can be substituted with one or more halogens; C1-C6straight or branched alkyl; hydroxy; C1-C6 straight or branched alkoxy;nitro; nitrile; unsubstituted amine or amine substituted with one ormore C1-C6 straight or branched alkyls; C1-C6 straight or branchedalkylcarbonyl or 5-membered or 6-membered heterocycloalkyl comprisingheteroatoms selected from the group consisting of N, O, and S; X is NHor O, provided that when R³ is unsubstituted or substituted aryl X is O;and n is an integer of 1-5.
 2. The disubstituted adamantyl derivativerepresented by formula 1 or a pharmaceutically acceptable salt thereofaccording to claim 1, wherein: R¹ is —X—(CH₂)_(n)—R³; R² is H or C1-C4straight or branched alkyl; R³ is unsubstituted or substituted phenyl,unsubstituted or substituted pyridine, pyrazine, imidazole, thiophene,benzothiophene, furan, or benzofuran, wherein the substituted phenyl orthe substituted pyridine, pyrazine, imidazole, thiophene,benzothiophene, furan or benzofuran is optionally substituted with oneor more fluoro, bromo, chloro, methyl, ethyl, propyl, isopropyl, butyl,hydroxy, t-butyl, methoxy, ethoxy, propoxy, butoxy, nitro, nitrile,amine, methylamine, dimethylamine, ethylamine, diethylamine, acetyl,ethylcarbonyl or 5-membered or 6-membered heterocycloalkyl comprisingheteroatoms selected from the group consisting of N, O, and S; X is NHor O, provided that when R³ is unsubstituted or substituted aryl X is O;and n is an integer of 1-3.
 3. The disubstituted adamantyl derivativerepresented by formula 1 or a pharmaceutically acceptable salt thereofaccording to claim 1, wherein: R¹ is —X—(CH₂)_(n)—R³; R² is methyl; R³is unsubstituted or substituted phenyl, or unsubstituted or substitutedfuran, wherein the substituted phenyl or the substituted heteroaryl isoptionally substituted with one or more chloro, bromo, methyl, ethyl,hydroxy, methoxy, ethoxy, ethylamine, acetyl, piperidine, piperazine,pyrolidine, tetrahydrofuran, or tetrahydrothiophene; X is NH or O,provided that R³ is unsubstituted or substituted phenyl X is O; and n isan integer of 1-3.
 4. The disubstituted adamantyl derivative representedby formula 1 or a pharmaceutically acceptable salt thereof according toclaim 1, wherein the disubstituted adamantyl derivative is: (1)methyl-3-(2-(4-(3-((furan-2-ylmethoxy)carbonyl)adamantane-1-yl)phenoxy)acetamido)benzoate;or (2)methyl-3-(2-(4-(4-((3,4-dimethoxybenzyloxy)carbonyl)adamantane-1-yl)phenoxy)acetamido)benzoate.5. A method for preparing a disubstituted adamantyl derivativerepresented by formula 1 comprising reacting a compound represented byformula 2 with a compound represented by formula 3 to give a compoundrepresented by formula 1, as shown in the below reaction formula 1:

wherein R¹ and R² are the same as defined in claim
 1. 6. Apharmaceutical composition for the inhibition of cancer metastasiscomprising a disubstituted adamantyl derivative represented by formula 1or a pharmaceutically acceptable salt thereof as an active ingredient:

wherein R¹ and R² are the same as defined in claim
 1. 7. Thepharmaceutical composition for the inhibition of metastasis according toclaim 6, wherein the disubstituted adamantyl derivative or thepharmaceutically acceptable salt thereof characteristically inhibits theexpression of Twist gene via suppressing accumulation of HIF-1α.
 8. Thepharmaceutical composition for the inhibition of metastasis according toclaim 6, wherein the cancer is solid cancer.
 9. The pharmaceuticalcomposition for the inhibition of metastasis according to claim 8,wherein the solid cancer is one or more selected from the groupconsisting of colorectal cancer, liver cancer, stomach cancer, breastcancer, colon cancer, bone cancer, pancreatic cancer, head & neckcancer, uterine cancer, ovarian cancer, rectal cancer, esophagealcancer, small bowel cancer, anal cancer, colon cancer, fallopian tubecarcinoma, endometrial carcinoma, uterine cervical carcinoma, vaginalcarcinoma, vulval carcinoma, Hodgkin's disease, prostate cancer, bladdercancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelviccarcinoma, and central nervous system tumor.
 10. A pharmaceuticalanticancer composition comprising a disubstituted adamantyl derivativerepresented by formula 1 or a pharmaceutically acceptable salt thereofas an active ingredient.

wherein R¹ and R² are the same as defined in claim 1.